Biological organisms are constantly required to prevent and repair damage to the genetic code. Damage to the genetic code is intimately related to cellular dysfunction, but there are several levels of defense against modification by both endogenous and exogenous sources of genotoxicity. Once covalent modification of the genetic code takes place the cell can respond with an arsenal of repair mechanisms including lesion specific DNA glycosylases, transferases, nucleotide/base-excision repair, and mismatch repair pathways. If the lesion is not removed from the DNA prior to replication then enzymes known as DNA polymerases will encounter the damage, and this encounter will, to a large extent, determine whether the damage will yield a permanent mutation in the genetic code. There are several types of polymerase in every cell. Some polymerases maintain a high degree of accuracy when copying DNA and are the main enzymes involved in replication of the genetic code. These so-called replicative polymerases are often less able to bypass damaged DNA because each step in catalysis has stringent molecular checkpoints that must be met before DNA synthesis occurs. Other polymerases possess lesion bypass abilities that can aid the replication fork when it encounters damage, but how these two general types of polymerases are coordinated in response to DNA damage remains unclear. Structural approaches including x-ray crystallograpy and hydrogen-deuterium exchange mass spectrometry will be combined with kinetic analysis in an effort to determine how two human Y-family DNA polymerases interact with the Werner syndrome protein during bypass of damaged DNA. Werner syndrome is characterized by premature aging and genomic instability that leads to unusually high incidence of sarcomas and mesenchymal tumors. Understanding how the enzymes that make our DNA interact when they encounter damage to our genetic code is an important part of understanding why certain chemicals are more toxic than others, how cancer develops, and even relates to why we age? The goals of our research proposal seek to answer some important questions related to how different types of "DNA making" enzymes function together and better define when the inherent functions of these enzymes fail to overcome the damage laid before them. Relevance: The proposal is relevant to our global understanding of genmoic maintenance. Genomic instability a thought to be a central feature of the normal aging process and during tumor development. A more detailed understanding of these processes can lead to better cancer therapeutics and possibly agents that improve the overall well-being of individuals as they age.